Synthesis of Drug Conjugates Via Reaction With Epoxide-Containing Linkers

ABSTRACT

The present invention relates to drug derivatives and linkers. The invention specifically relates to compounds and methods of phosphonates and linkers, that are useful as carriers for imaging agents and useful in the treatment of various bone diseases.

FIELD OF THE INVENTION

The present invention relates to compounds and methods of phosphonatesand linkers.

BACKGROUND OF THE INVENTION

Imaging agents incorporating a targeting drug and visualizing moiety areindispensable in medical diagnostics and are invaluable aids inpharmacological drug development (9, 10, 23). Fluorophores absorbing inthe visible region and emitting in the visible and near-infrared (IR)have found increasing application in this area owing to their scanningaccessibility, convenience of use and sensitivity to detection (29). 5-or 6-Carboxyfluorescein (5-FAM, 6-FAM) and other fluorescent visible andnear-IR labels are typically conjugated to a drug or protein by directreaction of a primary amine of the drug or protein with a label'sactivated group (e.g. succinimidyl esters (SE), isothiocyanates (ITC),sulfosuccinimidyl esters (SSE), tetrafluorophenyl esters (TFP),sulfodichlorophenol esters (SC), etc.), thus forming an amide bondbetween the label and drug/protein (14). However, in cases when theparent drug structure lacks a primary amine group, often a linker (3)between the drug and label or structural modification to the drug (27)is necessary for labeling. In general, amido links are preferable toesters which may be labile to hydrolysis in vitro or in vivo.

Bone-targeting nitrogen-containing bisphosphonate drugs (N-BPs) such as(1-hydroxy-2-pyridin-3-ylethane-1,1-diyl)bis(phosphonic acid) 1,[hydroxy(1H-imidazol-1-yl)methylene]bis(phosphonic acid),{1-hydroxy-3-[methyl(pentyl)amino]propane-1,1-diyl}bis(phosphonic acid),(3-amino-1-hydroxypropane-1,1-diyl)bis(phosphonic acid), and(4-amino-1-hydroxybutane-1,1-diyl)bis(phosphonic acid) are extensivelyused in the clinic to treat osteoporosis and other disorders of bonemetabolism (20, 21). Some bisphosphonate drugs have been shown toinhibit metastasis in bone cancer, and also to exhibit ananti-neoplastic effect on bone tumors (2, 7, 19).Alkylidenebisphosphonate drugs α-substituted with an aminoalkyl orN-containing heterocyclic group have been shown to inhibit specificallyone or more enzymes of the mevalonic pathway; in at least some cases,the nitrogen atom is sufficiently basic to be protonated atphysiological pH. X-ray crystallographic and modeling studies suggestthat interaction of this nitrogen with target enzyme active sitemoieties contributes significantly to inhibitory potency and thus to theefficacy of this class of anti-osteoporotic drugs (5, 8, 11, 12, 15,18). In contrast, the bone affinity is almost solely determined by thebisphosphonate moiety itself (18, 21, 28). Bisphosphonates have thegeneral structure:

Fluorescently labeled bisphosphonate drugs can be useful in improvingunderstanding of drug bone distribution, cellular distribution, and cellabsorption selectivity. The clinically significant but thus far poorlyunderstood anti-metastatic and anti-tumor cell effects of somebisphosphonates offers a further rationale for developing such imagingprobes. Recent reports of a small number of previously unidentifiedosteonecrotic onsets that may be linked to prolonged therapy with atleast one bisphosphonate also suggest an urgent requirement for improvedunderstanding of bisphosphonate drug distribution in bone tissues (17,22).

A conjugate of (4-amino-1-hydroxybutane-1,1-diyl)bis(phosphonic acid)with a near-IR fluorophore (Alexa Fluor® 488, commercially availablefrom Molecular Probes, Inc.), attached to the drug by formation of acarboxamide link with the drug's ε-amino group, was recently describeddrug (25). In this process, formation of the amide link greatly reducesthe basicity of the N atom, abolishing its ability to acquire a positivecharge by protonation. A comparable acylation approach for conjugatingheterocyclic N-BPs such as 1 (via its pyridine nitrogen) is not facile,and no fluorescently N-labeled versions of such compounds have beenavailable to date, to the inventors' knowledge. The structure of1-hydroxy-2-pyridin-3-ylethane-1,1-diyl)bis(phosphonic acid 1 is:

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to phosphonate drug or compoundconjugates that may be used to study bone diseases.

In another embodiment, the invention relates to phosphonate drug orcompound conjugates that may be used to study distribution of such drugsin bone tissues and cells.

In another embodiment, the invention relates to phosphonate drug orcompound conjugates that may be used to treat bone diseases.

In a related embodiment, the invention relates to phosphonate drug orcompound conjugates that may be used to detect bone diseases.

In accordance with one embodiment, the invention relates to methods ofsynthesizing phosphonate drug or compound conjugates that may be used tostudy bone diseases.

In accordance with another embodiment, the invention relates to methodsof synthesizing phosphonate drug or compound conjugates that may be usedto study distribution of such drugs in bone tissues and cells.

In accordance with another embodiment, the invention relates to methodsof synthesizing phosphonate drug or compound conjugates that may be usedto treat bone diseases.

In accordance with a related embodiment, the invention relates tomethods of synthesizing phosphonate drug or compound conjugates that maybe used to detect bone diseases.

In a closely related embodiment, the invention relates to methods ofusing phosphonate drug conjugates to study bone diseases.

In another closely related embodiment, the invention relates to methodsof using phosphonate drug or compound conjugates to study distributionof such drugs in bone tissues and cells.

In another closely related embodiment, the invention relates to methodsof using phosphonate drug or compound conjugates to treat bone diseases.

In yet another closely related embodiment, the invention relates tomethods of using phosphonate drug or compound conjugates to detect bonediseases.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the term “bone disease” refers to or describes anyaffliction that involves the skeletal system and encompasses anycondition that is associated with an impairment of the normal state ofthe skeletal system including congenital defects, pathologicalconditions such as cancer, and responses to environmental factors andinfectious agents (bacterial, viral, etc.). Examples of bone diseasesinclude but are not limited to osteoporosis, Paget's disease, metastaticbone cancers, hyperparathyroidism, rheumatoid arthritis, algodystrophy,stemo-costoclavicular hyperostosis, Gaucher's disease, Engleman'sdisease, disorders of bone metabolism, and the like.

The term “phosphonate” describes organic compounds containing one ormore C—PO(OH)₂ or C—PO(OR)₂ (with R=alkyl, aryl) groups. The“phosphonate” as used herein preferably refers to analogs ofphosphonate. Examples of phosphonates include but are not limited tobisphosphonates, phosphonoacetates, methylenebisphosphonates,phosphonocarboxylates, nitrogen-containing bisphosphonates, and thelike.

Phosphonates are preferably fluorescently labeled or conjugated withfluorophores, visible or near-infrared imaging agents. Examples offluorophores or near-infrared imaging agents include but are not limitedto Alexa Fluor dyes, Cye dyes, IRDyes, other fluorophores, near-infraredimaging agents, and the like. More specifically, fluorophores refer to5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), AMCA-X,Rhodamine Red-X, and the like, and near-infrared agents refer to AlexaFluor 647, and the like.

Fluorescently labeled phosphonates may be used to improve theunderstanding of drug bone distribution, cellular distribution, and cellabsorption selectivity.

The term “linker” refers to the moiety between the phosphonate andanother compound or structural modification to the phosphonate thatallows for conjugation of the phosphonate. Phosphonates may be linked tocompounds, imaging agents, structures, or other moieties that may beused in drug delivery. Examples include but not limited to drugs, beads,fluorescent labels, and the like.

Furthermore, phosphonate conjugates or compounds may used in a varietyof ways. For example, conjugates or compounds may be used as a drug forthe treatment of bone diseases or as a diagnostic for the detection ofbone disease. Conjugates or compounds can also be used to study bonedisease and the distribution of phosphonates in bone tissues, and bonecells.

Fluorescently labeled or conjugated phosphonates or compounds of theinvention are formulated to be compatible with its intended route ofadministration. Examples of routes of administration include parenteral,e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates; and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringes,or multiple dose vials made of glass or plastic.

In one embodiment, the compounds are prepared with carriers that willprotect the compounds against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Therapeutic agents, may also comprise siRNAsconjugated to cationic polypeptides, amphipathic compounds, polycations,liposomes or PEGylated liposomes. Biodegradable, biocompatible polymerscan be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.“Dosage unit form,” as used herein, refers to physically discrete unitssuited as unitary dosages for the subject to be treated, each unitcontaining a predetermined quantity of an active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The dosage required for treating a subject depends on the choice of theroute of administration, the nature of the formulation, the nature ofthe subject's illness, the subject's size, weight, surface area, age,and sex, other drugs being administered, and the judgment of theattending physician. Wide variations in the needed dosage are to beexpected in view of the variety of compounds available and the differentefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization as is well understood in the art. Encapsulation of thecompound in a suitable delivery vehicle (e.g., polymeric microparticlesor implantable devices) may increase the efficiency of delivery,particularly for oral delivery.

To practice methods of treatment, fluorescently labeled phosphonatecompounds are administered to a human or other mammal in need thereof atherapeutically effective amount of the compound. Indicationsappropriate to such treatment include bone diseases that include but arenot limited to osteoporosis, Paget's disease, metastatic bone cancers,hyperparathyroidism, rheumatoid arthritis, algodystrophy,stemo-costoclavicular hyperostosis, Gaucher's disease, Engleman'sdisease, disorders of bone metabolism, and the like.

To practice methods relating to the study of bone disease ordistribution of phosphonates in bone tissue or cells, fluorescentlylabeled compounds can be used in various model systems, includingenzymatic and cellular assays as well as in vivo. For enzymatic studies,fluorescent compounds can be pre-incubated with enzyme, and the reactionproducts can be detected according to standard procedures. For imagingof compounds of distribution in cells, fluorescent compounds can beadded to cell culture medium using standard methods known to thoseskilled in the art, and then visualized by methods such as confocalmicroscopy. For in vivo studies, compounds incorporating a near-IRimaging agent may be administered intravenously or by other appropriatemeans to the animal and subsequently visualized with MR fluorescenceimaging systems. Alternatively, compounds containing fluorescent labelsmay be administered and the distribution of the compound in bone tissuesor organs determined postmortem.

To practice the methods relating to methods of synthesizingfluorescently labeled analogs of phosphonates, the synthesis of1-(3-amino-2-hydroxypropyl)-3-(2-hydroxy-2,2-diphosphonoethyl)pyridiniumtrifluoroacetate 6 may be used as an example. The linking strategy iscentered on the reaction of N-t-BOC protected 1,2-epoxy-3-aminopropane 4to the nitrogen of the pyridine ring of 1 (Scheme 1). The low solubilityof 1 in organic solvents limits any reaction involving this compound toaqueous environments; however, “linker” 4 is highly soluble in nonpolarsolvents. Surprisingly, the N-alkylation reaction occurs with highregioselectivity and mild reaction conditions (aqueous conditions, pH5-6, 35-45° C.), unlike the previously reported O-alkylation of abisphosphonic acid with diethyloxiranylmethylamine (aqueous conditions,near neutral pH, 60-70° C.) (6). Surprisingly, at this temperature, theinventors' reaction yields only 10% O-alkylation and 90% N-alkylationand at 40° C., the reaction proceeds to nearly quantitative yield toafford the N-alkylated product 5 with less than 1% O-alkylation.

Intermediate 5 then undergoes subsequent deprotection with TFA to affordnovel compound 6 as a trifluoroacetate salt. This invention not onlyintroduces a primary amine to the parent drug for facile conjugation toactivated groups of fluorescent labels or other conjugate partners, butalso generates a permanent positive charge on the pyridinium nitrogenand introduces an additional hydroxyl group, which may increase thedrug's hydrophilicity. Additionally, by avoiding conjugation of thelinker group via the phosphoryl oxygens, the new compounds can retainbone affinity.

Analog 6 is then easily coupled to a group susceptible to reaction witha primary amine, for example the succinimidyl ester of5(6)-carboxyfluorescein (5(6)-FAM, SE, 7) under appropriate conditions(Scheme 2).

Several purification methods may be utilized to isolate the product,including size exclusion chromatography, reverse-phase HPLC, and TLC onsilica gel.

For example, Sephadex G10 columns eluted with TEAAc buffer can be usedto remove low molecular weight impurities, and the first eluting orangeband of the product collected. To ensure complete separation from bothfree label and unlabeled drug, a second purification step may be used,such as reverse-phase HPLC which was used to obtain pure compound 8.Although this purification sequence granted the purified product, poorseparation between the undesired compounds and the inventors' productduring size-exclusion chromatography lowered product yield.

Therefore, to remove free label, the inventors also developed a facilepurification method by TLC with 100% MeOH as the eluant. Extraction ofthe product from silica is performed with water as the solvent. Althoughthis method may not fully extract all of the desired compound fromsilica, it is rapid and convenient. NMR spectra of the extracts onlyshowed broad peaks, but treatment with Chelex to remove any traces ofmetal co-extracted from the silica, resulted in NMR spectra of goodquality that well characterized the structure and purity of the product.

To remove unlabeled 6, reverse-phase HPLC may be used advantageously,the unlabeled drugs having much shorter retention times than theproduct. Additionally, the 5- and 6-isomers (9 and 10, respectively) ofthe labeled product can be separated by HPLC, a method more costeffective than directly synthesizing these products from theirrespective isomerically pure fluoresceins purchased commercially.

Triethylammonium acetate is an appropriate buffer for such HPLCseparations. However, the higher volatility of triethylamine versusacetic acid causes a drop in pH, resulting in pH 4-5, when removing thebuffer under vacuum. The inventors found that, especially for largerscale purifications, the desired compounds tend to precipitate underthese conditions since they are much more water-soluble in neutral tobasic pH. Although the acetate buffer appears to be satisfactory inpreparation of smaller amounts of compound, the inventors haveidentified triethylammonium carbonate as a more suitable buffer formaintaining a basic pH and thus avoiding unwanted precipitation of thedesired product.

This same synthetic strategy was applied to two analogs of 1:2-hydroxy-2-phosphono-3-pyridin-3-ylpropanoic acid 11, aphosphonocarboxylate analog of 1 where one phosphonate moiety isreplaced with a carboxyl group, and(2-pyridin-3-ylethane-1,1-diyl)bis(phosphonic acid) 12, where theα-hydroxy is replaced with H (Scheme 3). Isomers of 11 and 12 may alsobe separated by HPLC although in this case, we chose to synthesize pureisomers of 11 from isomerically pure starting materials. All FAM-labeledcompounds have been characterized by high resolution mass spectrometryand ¹H and ³¹P NMR, UV absorption, and fluorescence emission spectra.The structures for 11 and 12 are:

UV spectra (taken on a DU 800 spectrophotometer) of all FAM-labeledcompounds (8-10 and 17-20) exhibit similar spectra to 5(6)-FAM, whichhas a reported ε₄₉₂=73,000 M⁻¹ cm⁻¹ at pH 7.2 (13, 16). However, thereis about a 35% larger ε at 260 nm due to a contribution from thepyridinium chromophore (ε=14,427 M⁻¹ cm⁻¹ at 262 nm) (24).

Fluorescence emission spectra (taken on a Jobin Yvon Horiba Fluoro Max-3fluorometer, with excitation wavelength at 490 nm and maximum emissionat ˜520 nm) of the FAM-labeled compounds typically show a loss of 10-20%of fluorescence intensity. Although care was given to ensure minimallight exposure while working with all labeled products, the slightdecrease in fluorescence may be due to some bleaching during work-up, ormay be an inherent characteristic of the FAM-labeled compounds. Forimaging purposes, the decrease does not affect the utility of thecompounds.

To study the stability of 8, the compound and 5- and 6-FAM, all in 0.1 Nphosphate buffer (pH 7.2), were stored in a freezer and in darkness forone week. Both solutions were analyzed by TLC on silica gel with 100%methanol as the eluant. The inventors' compound showed only one spot atthe baseline (UV detection at 365 nm), which corresponds to desiredproduct 8. The presence of 5(6)-FAM (which does not remain on thebaseline under these TLC conditions but rather travels quickly with thesolvent) was not detected in the solution containing compound 8, thusindicating that 8 is stable and does not hydrolyze to release freelabel. In addition, the same solutions were kept for an additional 6days at room temperature and no change was seen by analytical TLC. Smallaliquots of the phosphate solutions were also diluted in two otherbuffers: 50 mM HEPES (pH 7.0) and 50 mM TRIS (pH 7.7). The resulting newbuffer solutions were then kept at room temperature overnight andanalyzed by TLC. No decomposition of the inventors' compound wasobserved.

Chen et al. previously synthesized a fluorescent probe for proteins,derived from R-glycidol and 5-FAM, SE and 5(6)-FAM, SE (4). Then, thefluorescently labeled epoxide (in slight excess) reportedly reacts witha specific histidine residue of their target protein in high yields (4).In contrast, the inventors' approach involves the opening of the epoxideby the heterocylic nitrogen first, followed by subsequent conjugation tothe fluorescent label. This method may be more cost efficient thansynthesizing labeled epoxides, especially in cases involving moreexpensive commercially available imaging agents. Moreover, thepreviously reported method utilizes an ester bond to connect the epoxidemoiety to the rest of the probe, which is more labile to hydrolysisunder physiological conditions than an amide bond formed in the presentsynthesis. Finally, this method did not involve phosphonates attached toa heterocyclic nitrogen group.

Compounds 1, 11, and 12 may be labeled with the mixture of fluoresceinisomers and also using isomerically pure 7, Compounds 8-10 fluorescewith a green color that appears very similar to the green fluorescenceemitted by 7. In order to study compounds 1, 11, and 12 within the samebiological assay, each compound can advantageously be labeled withdifferently colored fluorescent emitters. For example, 11 and 12 can belabeled with the following fluorescent labels: Rhodamine Red-X (RhR-X)and 6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic acid (AMCA-X).The fluorescent color of RhR-X is red-orange while AMCA-X is blue. Bylabeling each drug with a different colored label, such as labeling 11with AMCA-X and 12 with RhR-X, the new imaging probes may besimultaneously visualized with FAM-labeled 8 within the same biologicalassay. Alternatively, the effect of the label on the properties of thedrug can be ascertained by preparing differently labeled versions of thesame drug. Many other useful applications of the invention may be made.For example, drugs that may not be susceptible to this labeling method,such as phosphonate compounds lacking a linkable nitrogen, may beindirectly imaged by their ability to displace a labeled phosphonatecompound from a site of binding such as bone. Compounds 23, 24, and 25can be synthesized and purified by methods similar to those describedfor FAM-labeled compounds (Scheme 4-5).

The Linking Strategy

To form the stable amido linkage to the FAM label, a terminal oxiranylamine is required. For this purpose, the corresponding allylamine 2 maybe first converted to the N-t-BOC protected compound 3. Epoxidation withm-chloroperbenzoic acid provides the oxirane 4. 1 was dissolved in waterand the pH adjusted to 6.4 with 1 N NaOH. This solution was combinedwith 4 in MeOH, yielding 5, which was then deprotected with TFA, giving6. To commercially available 5(6)-FAM, SE (Sigma Aldrich) in fresh andanhydrous DMF was added 6 in NaHCO₃ (pH 8.3-8.5) and the reactionmixture stirred at room temperature for 3 hours. The reaction mixturecan also be analyzed conveniently by silica gel TLC with UV illumination(100% MeOH), the fluorescent labeled product being easilydistinguishable (Rf 0.0) from 5(6)-FAM (Rf 1.0), a side product from thehydrolysis of the activated ester form. After removal of DMF and waterunder vacuum and redisolution in 50 mM TEAAc (pH 9), the crude reactionmixture was eluted through a G-10 Sephadex column (3 cm×40 cm) with 50mM TEAAc (pH 9) to remove traces FAM isomers. Each fraction eluted isanalyzed by silica gel TLC according to the method described above. Allfractions not containing 5(6)-FAM by this analysis method are thenadditionally purified and separated into its individual isomercomponents by preparative reverse-phase HPLC with the followingconditions: Dynamax C-18 column, flow rate 8.0 mL/min of 10% MeOH in 0.1N TEAAc (pH 7) to 40% of 75% MeOH in 0.1 N TEAAc (pH 7) in 12 min,increasing to 70% of 75% MeOH in 0.1 N TEAAc in 100 min, UV detection at260 nm. 9 (elution at 27 min) and 10 (elution at 44 min) are collectedseparately, dried, and isolated as a stable, reddish-orange solids,readily soluble in water and stable under neutral conditions and at roomtemperature for at least 24 hours.

A wide range of imaging agents, such as fluorescent and near-IR labelscommercially available as activated esters, may be incorporated into ourinvented compounds. These activated esters will allow a person ofordinary skill in the art to easily conjugate our linker compounds toany desired label. For example, Alexa Fluor® 647, succinimidyl ester 26(31), a near-IR label, was successfully conjugated to 6 yieldingcompound 27, purified by HPLC, and characterized by mass spectrometryand NMR, UV absorption, and fluorescence emission spectra (Scheme 6).

The N-alkylation to form a pyridinium ring through this epoxidetechnology is a highly effective approach that gives excellentregioselectivity and yields, providing pure materials. This chemistrymakes possible a general synthesis of previously unavailablefluorescently labeled N-BPs. Thus, it is applicable to other types ofnitrogen besides pyridinyl nitrogens, such as the tertiary nitrogens in{1-hydroxy-3-[methyl(pentyl)amino]propane-1,1-diyl}bis(phosphonic acid)(28), and nucleophilic nitrogens in other heterocyclic compounds, suchas that in [hydroxy(1H-imidazol-1-yl)methylene]bis(phosphonic acid) (29)or the in the bicyclic structure found in(1-hydroxy-2-imidazo[1,2-a]pyridin-3-ylethane-1,1-diyl)bis(phosphonicacid) (30). The structures of 28, 29, and 30 are:

This approach is not restricted only to N-bisphosphonates, as alreadydemonstrated by the synthesis of a fluorescent phosphonocarboxylate (PC)analog (17-19 and 24), but may also be applied for any type of compoundcontaining the mildly nucleophilic heterocyclic nitrogens or tertiarynitrogens. In cases where such cyclic systems are absent but aphosphorus-oxygen (P—O) bond is still present in the parent drug, theconditions of this epoxide chemistry may be altered to favorO-alkylation, thus generating the linker through the P—O bond.Additionally, this technology may be applied to similar BP and PCcompounds with no known pharmacological activity in cases where anactive drug may not be needed. Thus, the invention may be applied to anextensive range of compounds (including BPs, PCs, otherphosphorus-containing compounds, and compounds with tertiary nitrogensor nitrogen-containing heterocycles) to introduce a “linker” moietynecessary for direct acylation or other compatible conjugation reactionwith any other groups, including imaging agents or other moieties suchas drugs requiring or benefiting from delivery to bone.

The following examples are intended to illustrate, but not to limit, thescope of the invention. While such examples are typical of those thatmight be used, other procedures known to those skilled in the art mayalternatively be utilized. Indeed, those of ordinary skill in the artcan readily envision and produce further embodiments, based on theteachings herein, without undue experimentation.

EXAMPLES Synthesis of tert-Butyl N-allylcarbamate (3)

Tert-butyl N-allylcarbamate was synthesized according to the method ofRocheblave (30). 2.3 mL (30 mmol, 1 equiv) of freshly distilledallylamine 2 in 10 mL CH₂Cl₂ was cooled in an ice bath (0° C.). To thiscold solution was added 6.54 g Boc₂O (30 mmol, 1 equiv) in 20 mL CH₂Cl₂.The solution was brought to room temperature and stirred for 4 hours.

The reaction mixture was then diluted with additional CH₂Cl₂ and washedwith 5% citric acid solution followed by brine. The organic layer wasdried over Na₂SO₄ and concentrated in vacuo, yielding 3.29 g (68% yield)of 3.

¹H NMR (500 MHz, CDCl₃): δ1.38 (s, 9H), 3.68 (brs, 2H), 4.53 (brs, 1H),5.02-5.16 (m, 2H), 5.72-5.84 (m, 1H).

Synthesis of tert-butyl N-(2-oxiranylmethyl)carbamate (4)

Tert-butyl N-(2-oxiranylmethyl)carbamate was synthesized according tothe method of Rocheblave (30). 1 g (6 mmol, 1 equiv) of N-t-BOCprotected 3 was dissolved in 50 mL dry CH₂Cl₂. The solution was broughtto 0° C. and kept cold upon addition of 2.8 g (12 mmol, 2 equiv) MCPBA.The solution was then brought to room temperature and stirred overnight.

About half of the reaction mixture was taken and diluted with additional˜80 mL of CH₂Cl₂. The solution was washed with 10% Na₂SO₃, followed bywashing with saturated NaHCO₃ 3 times, and finally by washing withwater. The organic layer was dried over Na₂SO₄ and concentrated invacuo, yielding crude epoxide 4. By ¹H NMR, approximately 85% yield wasachieved.

¹H NMR (400 MHz, CDCl₃): δ 1.44 (s, 9H), 2.59-2.78 (brm, 2H), 3.04-3.54(m, 3H), 4.75 (brs, 1H).

General Synthesis of N-Alkylated Drug Analogs

The parent drug is dissolved in water and pH is adjusted to ˜6 with 1 NNaOH. Epoxide 4 is dissolved in minimal MeOH, and the reaction mixtureis heated at 35-50° C. for 18-45 hours. Upon addition of the methanolsolution to the water solution, slight precipitation occurs. Solubilityis increased with slight heating and as reaction progresses. Thereaction is monitored by ³¹P NMR and can also be monitored by analyticalreverse-phase HPLC. After 90-95% of the desired product is obtained, thesolvent is removed in vacuo, and the resulting white powder is washedwith diethyl ether, filtered and dried. Standard deprotection isperformed with TFA. After the reaction mixture is stirred for 3-4 hoursat room temperature, the solvent is removed in vacuo, and the resultingcrystals are washed with diethyl ether and methanol to yield theappropriate drug-linker analog, used without additional purification,for labeling reactions.

Synthesis of1-(3-amino-2-hydroxypropyl)-3-(2-hydroxy-2,2-diphosphonoethyl)pyridiniumtrifluoroacetate (6)

287 mg of monosodium salt of 1 (0.9 mmol, 1 equiv) was dissolved in 4 mLwater and pH adjusted to 6.2 with 1 N NaOH. 163 mg of 4 (0.9 mmol, 1equiv) in minimal MeOH was added. Slight precipitation upon additionoccurs, but will slowly disappear as reaction progresses. The reactionmixture was stirred at 40° C. for 18.5 hours, yielding 90% of 5 by ³¹PNMR. The solvent was removed in vacuo, and the remaining solids werewashed with ether, filtered, and dried in a dessicator. The remainingsolids 5 were then used without further purification.

¹H NMR (400 MHz, D₂O): δ 1.27 (s, 9H), 3.07-3.30 (m, 4H), 3.95-4.03 (m,1H), 4.18-4.27 (dd, J=13.7 Hz, 3.7 Hz, 1H), 4.58-4.65 (part. obscured byHDO, about 1H), 7.75 (t, J=6.8 Hz, 1H), 8.39 (d, J=7.8 Hz, 1H), 8.43 (d,J=6.5 Hz, 1H), 8.65 (s, 1H). ³¹P{¹H} NMR (400 MHz, D₂O): δ 16.33 (d,J=21.1 Hz, 1P), 16.55 (d, J=21.1 Hz, 1P).

The entire sample of 5 was dissolved in 3 mL water. 3 mL TFA was slowlyadded, and the solution was stirred at room temperature for 3 hours.According to NMR, 100% yield is achieved of 6. The solvent was thenremoved in vacuo, and the resulting solids were washed with ether,filtered, and dried, yielding 6 as white crystals. 6 was then usedwithout further purification.

¹H NMR (400 MHz, D₂O): δ 3.01 (t, J=11 Hz, 1H), 3.31 (d, J=12.6 Hz, 1H),3.39 (t, J=11.6 Hz, 2H), 4.25-4.33 (m, 1H), 4.36-4.44 (br, 1H), 7.88 (t,J=6.6 Hz, 1H), 8.48 (d, J=8.0 Hz, 1H), 8.58 (d, J=5.5 Hz, 1H), 8.77 (s,1H). ³¹P{¹H} NMR (500 MHz, D₂O): δ 16.04 (d, J=27.5 Hz, 1P), 16.40 (d,J=27.5 Hz, 1P).

Synthesis of1-(3-amino-2-hydroxypropyl)-3-(2-carboxy-2-hydroxy-2-phosphonoethyl)pyridiniumtrifluoroacetate (15)

0.52 g of 11 (2 mmol, 1 equiv) was dissolved in 10 mL water and pHadjusted to 5.9 with 1 N NaOH. 0.445 g of 4 (2.6 mmol, 1.3 equiv) inminimal MeOH was added. Slight precipitation upon addition occurs, butwill slowly disappear as reaction progresses. The reaction mixture wasstirred at 50° C. for 6 hours and then stirred at room temperatureovernight, yielding 90% of 13 by ³¹P NMR. The solvent was removed invacuo, and the remaining solids were washed with ether, filtered, anddried in a dessicator. The remaining solids 13 were then used withoutfurther purification.

¹H NMR (400 MHz, D₂O): 1.27 (s, 9H), 3.00-3.23 (m, 3H), 3.45 (dd, J=14.2z, 3.6 Hz, 1H), 3.91-4.00 (m, 1H), 4.19-4.27 (m, 1H), 4.58-4.64 (br,1H), 7.78 (dd, J=8.3 Hz, 6.2 Hz, 1H), 8.24-8.29 (m, 2H), 8.47 (d, J=6.0Hz, 1H), 8.49-8.53 (brd, 1H). ³¹P{¹H} NMR (400 MHz, D₂O): δ 14.96 (s,1P), 14.98 (s, 1P).

The entire sample of 13 was dissolved in 50:50 water:TFA. The solutionwas stirred at room temperature for 4 hours. According to NMR, 100%yield is achieved of 15. The solvent was then removed in vacuo, and theresulting solids were washed with ether, filtered, and dried, yielding15 as white crystals. 15 was then used without further purification.

¹H NMR (400 MHz, D₂O): δ 2.90 (m, 1H), 3.18-3.28 (m, 2H), 3.50 (m, 1H),4.14-4.22 (m, 1H), 4.31-4.40 (m, 1H), 4.71-4.74 (m, 1H), 7.87 (dd, J=8.3Hz, 6.1 Hz, 1H), 8.34-8.39 (brd, 1H), 8.56-8.68 (m, 2H). ³¹P{¹H} NMR(400 MHz, D₂O): δ 12.53 (s, 1P), 12.62 (s, 1P).

Synthesis of1-(3-amino-2-hydroxypropyl)-3-(2,2-diphosphonoethyl)pyridiniumtrifluoroacetate (16)

38 mg 12 (1.4 mmol, 1 equiv) was dissolved in 1 mL water and pH broughtto 5.4 with 1 N NaOH. To this solution was added 34 mg of 4 (1.8 mmol,1.2 equiv) in minimal MeOH. The reaction mixture was stirred at 40° C.overnight, and the reaction was followed by ³¹P NMR. After 19 hours, 20%of starting material 12 remained. Thus, an additional 7 mg of 4 in MeOHwas added to the reaction mixture. After 42 hours, 90% of the desiredproduct was obtained. The solvent was removed in vacuo, and theresulting white powder was washed with diethyl ether, filtered, anddried. The remaining solids 14 were used without further purification.

¹H NMR (400 MHz, D₂O): 1.26 (s, 9H), 2.12 (tt, J=21.2 Hz, 7.1 Hz, 1H),3.09-3.24 (m, 4H), 3.94-4.00 (m, 1H), 4.22 (dd, J=13.7 Hz, 9.6 Hz, 1H),4.57-4.64 (m. 1H), 7.84 (dd, J=8.6 Hz, 6.1 Hz, 1H), 8.40 (d, J=8.1 Hz,1H), 8.50 (d, J=6.1 Hz, 1H), 8.69 (s, 1H). ³¹P{¹H} NMR (400 MHz, D₂O): δ17.20-17.28 (m, 2P).

The entire sample of 14 was dissolved in water. An equal volume of TFAwas slowly added, and the solution was stirred at room temperature for 3hours. According to NMR, 100% yield is achieved of 16. The solvent wasthen removed in vacuo, and the resulting solids were washed with diethylether and methanol, filtered, and dried, yielding 16 as white crystals.16 was then used without further purification.

¹H NMR (400 MHz, D₂O): δ 2.18-2.36 (brt, 1H), 2.92 (brt, 1H), 3.14-3.28(m, 3H), 4.16-4.24 (m, 1H), 4.60 (dd, J=13.5 Hz, 9.5 Hz, 1H), 7.79 (dd,J=8.7 Hz, 6.1 Hz, 1H), 8.37 (d, J=8.2 Hz, 1H), 8.44 (d, J=6.1 Hz, 1H),8.64 (s, 1H). ³¹P{¹H} NMR (400 MHz, D₂O): δ 17.2-18.0 (m, 2P).

Synthesis of3-{3-[(tert-butoxycarbonyl)amino]-2-hydroxypropyl}-1-(2-hydroxy-2,2-diphosphonoethyl)-1H-imidazol-3-ium

9.8 mg of 29 was dissolved in 1 mL H₂O and pH adjusted to 7.4 withNa₂CO₃. To this solution was added 32.7 mg of 4 in minimal MeOH. Thereaction mixture was heated at 60° C. overnight, yielding compound 31 by³¹P NMR and ESI-MS.

³¹P NMR (400 MHz, D₂O): δ 14.11 (s). MS (negative ion ESI-MS, calculated446.1093 m/z, found 443.9 m/z).

Synthesis ofN-{3-[(tert-butoxycarbonyl)amino]-2-hydroxypropyl}-N-(3-hydroxy-3,3-diphosphonopropyl)-N-methylpentan-1-aminium

9.3 mg of 28 was dissolved in 1 mL H₂O and pH adjusted to 9.8 withNa₂CO₃. To this solution was added 27.1 mg of 4 in minimal MeOH. Thereaction mixture was stirred at 50-60° C. for 2 weeks, yielding compound32 by ³¹P NMR spectrum.

³¹P NMR (400 MHz, D₂O): δ 16.82 (s).

Synthesis of (8)

The following synthesis and purification steps were performed underminimal lighting. 177 mg of 6 (0.4 mmol, 5 equiv) was dissolved in 2 mLof 0.1 N NaHCO₃. The pH of the solution was adjusted to 8.3 with Na₂CO₃.41 mg of 5(6)-FAM, SE (7) (0.08 mmol, 1 equiv) was dissolved in 200 μLanhydrous DMF and then combined with water solution, forming a darkred-orange solution with small amount of precipitate. The pH was againadjusted to 8.1 with Na₂CO₃, causing the precipitate to dissolve, andthe reaction mixture was stirred at room temperature for 3 hours.

The reaction mixture was directly placed on TLC plates with 100% MeOH aseluant. Free label moves quickly with the solvent, resulting in a yellowupper band, while all phosphorus containing compounds remain on thebaseline, a dark orange band. The bottom band was extracted from thesilica with HPLC water and Chelex (sodium form). The solution wascentrifuged and concentrated in vacuo to yield dark red-orange solids.

The solids were dissolved in HPLC water and filtered through Nanosep 30KOmega filters. The solution was then purified by reverse-phase HPLC:Dynamax C18 (21.4 mm×25 cm) column, flow rate 8.0 mL/min, gradient asfollows: 10% MeOH in 0.1 N TEAAc (pH 7) to 40% of 75% MeOH in 0.1 NTEAAc (pH 7) in 12 min, increasing to 70% of Buffer B in 100 min. Majorpeaks eluting at 27 and 75 minutes were collected and combined. Uponremoval of buffer, product was precipitating out of solution. Thus, asecond HPLC purification was performed with the same conditions asbefore but with TEAC buffers (pH 7.5). This allowed for basic conditionswhen buffer was removed in vacuo. To help remove excess TEA, the HPLCpurified product was dissolved in water. To this solution was added ˜10excess of NaI in water. The solvent was removed in vacuo, yielding darkred solids. The solids were washed with acetone and centrifuged. Thesolids were then re-dissolved in water to help remove acetone and thesolvent removed in vacuo to yield 8 as red solids. The final amount of 8was calculated from 17V absorption spectra with ε=73000 M⁻¹ cm⁻¹ at pH7.2 and the compound was lyophilized.

8 (as disodium, monotriethylammonium iodide salt): ¹H NMR (400 MHz,D₂O): 3.33-3.39 (m, 2H), 3.43-3.66 (m, 2H), 4.08-4.42 (m, 3H), 4.72-4.80(brd, 1H), 6.36-6.48 (m, 4H), 6.93 (d, 2H), 7.09 (s, 1H), 7.43 (s,0.4H), 7.68-7.87 (m, 2H), 8.06 (s, 0.6H), 8.37-8.56 (m, 2H), 8.69-8.78(2 s, 1H). ³¹P{¹H} NMR (400 MHz, D₂O): δ 16.26 (d, J=23 Hz, 1P), 16.50(d, J=23 Hz, 2P).

Synthesis and Separation of (9 and 10)

11 mg of 6 (0.2 mmol, 1 equiv) was dissolved in 1 mL H₂O and 1.5 mL 0.1N NaHCO₃. The pH was adjusted to 8.4 with additional Na₂CO₃ to bring pHto 8.4. To this solution was added 100 mg of 7 (0.2 mmol, 1 equiv) in˜700 μL anhydrous DMF in darkness. After the addition of the label, thepH was again adjusted to 8.4 to increase solubility of the label. Thereaction mixture was stirred at room temperature in darkness for 3hours.

The solvent was then removed in vacuo, yielding bright reddish-orangesolids. The unpurified product was then re-dissolved in water andpurified by size exclusion chromatography with a Sephadex G10 column andeluted with 50 mM TEAAc. The bright orange compound can easily be seentraveling through the column. The first ˜20 mL of compound eluted wereanalyzed by TLC with 100% MeOH and showed no traces of 5(6)-FAM (Rf1.0).

The solvent of this fraction was then removed in vacuo and re-dissolvedin 10% MeOH of 0.1 N TEAAc buffer (pH 7). The compound was then purifiedby reverse-phase HPLC under the following conditions: Dynamax C18 (21.4mm×25 cm) column, flow rate 8.0 mL/min of 10% MeOH in 0.1 N TEAAc (pH 7)to 40% of 75% MeOH in 0.1 N TEAAcO (pH 7) in 12 min, increasing to 70%of 75% MeOH in 0.1 N TEAAc in 100 min, UV detection at 260 nm. 9 and 10eluted at very different retention times, 27 and 44 minutes,respectively; each isomer was collected separately. Each fractioncollected was analyzed by TLC (eluted with 100% MeOH) to ensure notraces of 5(6)-FAM was present. Each isomer was then concentrated invacuo to remove buffer. The final amount of 9 and 10 was calculated fromUV absorption spectra with ε=73000 M⁻¹ cm⁻¹ at pH 7.2. The compoundswere then lyophilized to yield bright reddish-orange solids.

9 (retention time of 44 minutes, as triethylammonium acetate salt): ¹HNMR (400 MHz, D₂O): δ 3.37 (brt, 2H), 3.57 (dd, J=14.1 Hz, 6.7 Hz, 1H),3.61-3.69 (m, 1H), 4.22-4.31 (m, 1H), 4.36-4.45 (m, 1H), 4.79 (d, 1H),6.61-6.71 (m, 4H), 7.11 (d, J=9.2 Hz, 2H), 7.33 (d, J=8.0 Hz, 1H), 7.56(s, 0.14H), 7.83 (t, J=7.2 Hz, 1H), 7.91 (d, J=8.1 Hz, 1H), 8.15 (s,1H), 8.44 (d, J=8.3 Hz, 1H), 8.56 (d, J=5.9 Hz, 1H), 8.74 (s, 1H).³¹P{¹H} NMR (500 MHz, D₂O): δ 16.47 (brs).

HRMS (positive ion MALDI gave the free acid molecular cation, calculated715.1089 m/z, found 715.1055 m/z (negative ion MALDI, low resolutionmass spectra, gave a major peak at 713).

10 (retention time of 27 minutes, as triethylammonium acetate salt): ¹HNMR (400 MHz, D₂O): δ 3.27-3.37 (m, 2H), 3.44 (dd, J=14 Hz, 6.9 Hz, 1H),3.58 (dd, J=14 Hz, 4.9 Hz, 1H), 4.19 (brs, 1H), 4.35 (dd, J=14 Hz, J=9.3Hz, 1H), 6.60-6.73 (m, 4H), 7.04 (d, J=9.4 Hz, 2H), 7.53 (s, 1H), 7.81(dd, J=8.2 Hz, 6.4 Hz, 1H), 7.88 (d, J=8.1 Hz, 1H), 7.95 (dd, J=8.0 Hz,1.6 Hz, 1H), 8.43 (d, J=8.1 Hz, 1H), 8.53 (d, J=6.5 Hz, 1H), 8.70 (s,1H). ³¹P{¹H} NMR (500 MHz, D₂O): δ 16.51 (brs).

HRMS (positive ion MALDI gave the free acid molecular cation, calculated715.1089 m/z, found 715.1082 m/z).

HRMS (positive ion MALDI gave the free acid molecular cation, calculated715.1089 m/z, found 715.1082 m/z).

Direct Synthesis of 9 from 5-FAM, SE

To first confirm isomer assignments, 9 was directly synthesized from5-FAM, SE according to the procedure directly above. The compound wasfirst purified by reverse-phase HPLC according to conditions describedabove, but no separation from free label was seen. The fluorescentcompounds were collected and additionally purified on a Sephadex G10column according to conditions above. ¹H and ³¹P NMR spectra wereexactly the same as 9 synthesized from 5(6)-FAM and isolated by HPLCseparation.

Synthesis of (17)

The following synthesis and purification steps were performed underminimal lighting. 158 mg of 15 (0.3 mmol, 3 equiv) was dissolved in 1 mLof water. The pH of the solution was adjusted to 8.3 with Na₂CO₃. 53 mgof 5(6)-FAM, SE (7) (0.1 mmol, 1 equiv) was dissolved in 200 μLanhydrous DMF and then combined with water solution, forming a darkred-orange solution with small amount of precipitate. The pH was againadjusted to 8.1 with Na₂CO₃, causing the precipitate to dissolve, andthe reaction mixture was stirred at room temperature overnight.

The reaction mixture was directly placed on TLC plates with 100% MeOH aseluant. Free label moves quickly with the solvent, resulting in a yellowupper band, while all phosphorus containing compounds remain on thebaseline, a dark orange band. The bottom band was extracted from thesilica with HPLC water and Chelex (sodium form). The solution wascentrifuged and concentrated in vacuo to yield dark red-orange solids.The solution was then purified by HPLC: Dynamax C18 (21.4 mm×25 cm)column, flow rate 8.0 mL/min, gradient as follows: isocratic elution of20% MeOH in 0.1 N TEAC (pH 7) for 12 min, linearly increasing to 100% of70% MeOH in 0.1 N TEAC (pH 7) in 22 min, UV detection at 260 nm. Peakseluting at 25-40 minutes were collected together as 17. The final amountof 17 was determined from UV absorption spectra (ε=73000 M⁻¹ cm⁻¹ at pH7.2), and the compound was lyophilized to yield bright red-orangesolids.

17 (as triethylammonium acetate salt): ¹H NMR (400 MHz, D₂O): δ3.27-3.62 (m, 3H), 4.03-4.40 (m, 2H), 6.42 (m, 4H), 6.92 (dd, J=9.5 Hz,3.5 Hz, 2H), 7.10 (d, J=8.1 Hz, 1H), 7.42 (s, 0.4H), 7.70-7.86 (m, 2H),8.06 (s, 0.6H), 8.27 (brs, 1H), 8.46-8.63 (m, 2H). ³¹P{¹H} NMR (400 MHz,D₂O): δ 15.28 (brs, 1P).

HRMS (positive ion MALDI gave the free acid molecular cation, calculated679.1335 m/z, found 679.1321 m/z).

Direct Synthesis of 18 and 19 from 5- and 6-FAM, SE, Respectively

Single isomers of 17 were synthesized directly from isomerically purestarting materials (5-FAM, SE or 6-FAM, SE) according to method aboveused for isomeric mixtures.

18 (retention time 28 minutes, as triethylammonium acetate salt): ¹H NMR(400 MHz, D₂O): δ 3.39-3.52 (m, 2H), 3.55-3.63 (m, 1H), 4.14-4.23 (m,1H), 4.30-4.40 (m, 1H), 6.43-6.60 (m, 4H), 7.04 (d, J=9.0 Hz, 2H), 7.25(d, J=8.2 Hz, 1H), 7.77-7.86 (m, 2H), 8.08 (s, 1H), 8.23-8.33 (brs, 1H),8.45-8.62 (m, 2H).

HRMS (positive ion MALDI gave the free acid molecular cation, calculated679.1324 m/z, found 679.1356 m/z).

19 (retention time 25 minutes, as triethylammonium acetate salt): ¹H NMR(400 MHz, D₂O): δ 3.31-3.45 (m, 2H), 3.47-3.55 (m, 1H), 4.05-4.15 (m,1H), 4.24-4.34 (m, 1H), 6.45-6.59 (m, 4H), 7.00 (d, J=9.0 Hz, 2H), 7.48(s, 1H), 7.71-7.80 (m, 2H), 7.85 (dd, J=8.1 Hz, J=1.7 Hz, 1H), 8.21-8.27(m, 1H), 8.47-8.55 (m, 2H).

HRMS (positive ion MALDI gave the free acid molecular cation, calculated679.1324 m/z, found 679.1321 m/z).

Synthesis of (20)

53 mg of 16 (0.1 mmol, 3.3 equiv) was dissolved in 0.5 mL HPLC water and0.5 mL of 0.1 N NaHCO₃. The pH was adjusted to 8.3 with Na₂CO₃. 18 mg of5(6)-FAM, SE (7) (0.03 mmol, 1 equiv) was dissolved in 100 μL anhydrousDMF and then combined with water solution, forming a dark red-orangesolution with small amount of precipitate. The pH was again adjusted to8.9 with Na₂CO₃, causing the precipitate to dissolve, and the reactionmixture was stirred at room temperature overnight.

The reaction mixture was purified by TLC with 100% MeOH as the eluant.The dark orange bottom band was extracted from silica with water andChelex (sodium form). The solution was centrifuged and solvent removedin vacuo, yielding dark red solids. The solids were dissolved in 20%MeOH in 0.1 N TEAAc buffer (pH 7) and filtered through Nanosep 30K Omegafilters. The solution was then purified by HPLC: Dynamax C18 (21.4 mm×25cm) column, flow rate 8.0 mL/min, gradient as follows: isocratic elutionof 20% MeOH in 0.1 N TEAC (pH 7) for 12 min, linearly increasing to 100%of 70% MeOH in 0.1 N TEAC (pH 7) in 22 min, UV detection at 260 nm.Peaks eluting from 27-45 minutes were collected as 20. The final amountof 20 was determined from UV absorption spectra (ε=73000 M⁻¹ cm⁻¹ at pH7.2), and the compound was lyophilized to yield red-orange solids.

20 (as triethylammonium acetate salt): ¹H NMR (400 MHz, D₂O): δ2.07-2.27 (m, 1H), 3.10-3.27 (m, 2H), 3.35 (dd, J=14.2 Hz, 6.8 Hz,0.4H), 3.44-3.53 (m, 1H), 3.60 (dd, J=14.2 Hz, 4.7 Hz, 0.6H), 4.08-4.16(m, 0.4H), 4.18-4.25 (m, 0.6H), 4.25-4.40 (m, 1H), 4.71-4.77 (m, 1H),6.40-6.52 (m, 4H), 6.96 (dd, J=8.1 Hz, 4.1 Hz, 2H), 7.17 (d, J=8.1 Hz,0.6H), 7.44 (s, 0.4H), 7.72 (d, J=8.1 Hz, 0.4H), 7.76-7.86 (m, 2H), 8.07(d, J=1.7 Hz, 0.6; H), 8.38 (t, J=8.7 Hz, 1H), 8.47 (d, J=5.9 Hz, 1H),8.53 (d, J=6.1 Hz, 1H), 8.67-8.71 (2 s, 1H). 31P{1H} NMR (400 MHz, D₂O):δ 16.51 (brs).

HRMS (positive ion MALDI gave the free acid molecular cation, calculated699.1139 m/z, found 699.1137 m/z).

Synthesis of (23)

11.2 mg of 6 was dissolved in 0.5 mL H₂O and pH adjusted to 9 withNa₂CO₃. To this solution was added 5 mg of RhR-X, SE 21 in 250 μL DMF,and the reaction mixture stirred overnight. The solvent was thenconcentrated under vacuum, and the resulting solids were dissolved inH₂O and purified by TLC (eluted with 100% MeOH). The band at the originwas extracted with H₂O, and the solution was centrifuged and solventremoved in vacuo. The solids are dissolved in H₂O, and the solution isthen purified by HPLC: Beckman Ultrasphere C18 (250×10 mm), flow rate6.0 mL/min, gradient as follows: isocratic elution of 20% MeOH in 0.1 NTEAC (pH 7) for 5 min, linearly increasing to 100% of 75% MeOH in 0.1 NTEAC (pH 7) in 6 min, UV detection at 260 nm. Peak eluting at 12 minuteswere collected as 23. The final amount of 23 was determined from UVabsorption spectra (ε=114850 M⁻¹ cm⁻¹ at pH 7.5).

23 (as triethylammonium carbonate salt): ¹H NMR (400 MHz, D₂O): δ 1.26(m, 2H), 1.38 (m, 2H), 2.10 (t, 2H), 3.01-3.09 (brm, 4H), 3.38-3.49(brm, 8H), 3.99 (m, 1H), 4.12 (m, 1H), 4.51 (1H), 6.73 (s, 2H), 6.79 (d,2H), 6.86. (d, 2H), 7.50 (d, 1H), 7.71 (t, 1H), 8.09 (d, 1H), 8.35-8.45(brm, 2H), 8.66 (s, 1H).

MS (positive ion ESI-MS gave the free acid molecular cation, calculated1011.2913 m/z, found 1008.28 m/z).

Synthesis of (24)

10.9 mg of 15 was dissolved in 0.5 mL of 0.1 N NaHCO₃ buffer, and pHadjusted to 8.7 with Na₂CO₃. To this solution was added 5 mg ofRhodamine Red-X, SE 21 in 500 μL DMF, and the reaction mixture stirredovernight. The solution was then placed on a TLC plate (7 cm×20 cm), andeluted 3× with MeOH. The bottom band was extracted with H2O, and thesolution was centrifuged and solvent removed in vacuo. The solids weredissolved in 20% MeOH in 0.1 N TEAAc buffer (pH 7) and filtered throughNanosep 30K Omega filters. The solution is then purified by HPLC:Beckman Ultrasphere C18 column (250×10 mm), flow rate 4.0 mL/min, UV at260 nm, gradient as follows: 20% MeOH in 0.1 N TEAC (pH 7.5) for 4 min,linearly increasing to 100% of 70% MeOH in 0.1 N TEAC (pH 7.5) in 19min, UV detection at 260 nm. The final amount of 24 was determined fromUV absorption spectra (ε=114850 M⁻¹ cm⁻¹ at pH 7.5).

24 (as triethylammonium carbonate salt): ¹H NMR (400 MHz, D₂O): δ 1.27(m, 2H), 1.39 (m, 2H), 2.09 (t, 2H), 2.89-2.99 (brm, 4H), 3.10-3.16(brm, 2H), 3.20-3.27 (brm, 1H), 3.38-3.49 (brm, 8H), 3.95 (m, 1H), 4.14(m, 1H), 4.51 (1H), 6.63 (s, 2H), 6.72 (d, 2H), 6.79. (d, 2H), 7.43 (d,1H), 7.73 (t, 1H), 8.08 (d, 1H), 8.25-8.30 (brm, 1H), 8.42 (d, 2H), 8.50(d, 1H). ³¹P NMR (500 MHz, D₂O): 15.11 (s).

HRMS (positive ion MALDI gave the free acid molecular cation, calculated975.3148 m/z, found 974.3118 m/z).

Synthesis of (25)

36.2 mg of 16 was dissolved in 3 mL 0.1 N NaHCO₃ with pH adjusted to 8.3with Na₂CO₃. 10 mg of AMCA-X, SE in 400 μL anhydrous DMF was added, andthe pH re-adjusted to 8.3 with Na₂CO₃. The reaction mixture was stirredin darkness at rt overnight. The reaction mixture was purified by TLCwith 100% MeOH as the eluant. The bottom band was extracted from silicawith water and Chelex (sodium form). The solution was centrifuged andsolvent removed in vacuo. The solids are dissolved in 20% MeOH in 0.1 NTEAAc buffer (pH 7) and filtered through Nanosep 30K Omega filters. Thesolution is then purified by HPLC: Dynamax C18 (21.4 mm×25 cm) column,flow rate 6.0 mL/min, gradient as follows: isocratic elution of 20% MeOHin 0.1 N TEAC (pH 7) for 12 min, linearly increasing to 100% of 70% MeOHin 0.1 N TEAC (pH 7) in 22 min, UV detection at 260 nm. Peaks elutingfrom 27-45 minutes were collected as 25. The final amount of 25 wasdetermined from UV absorption spectra (ε=16000 M⁻¹ cm⁻¹ at pH 7.5).

25 (as triethylammonium carbonate salt): ¹H NMR (400 MHz, D₂O): δ 1.36(m, 2H), 1.45 (m, 2H), 2.04-2.19 (m, 3H), 2.21 (d, 2H), 3.2-3.4 (brm,5H), 3.68-3.77 (m, 1H), 3.99-4.06 (brm, 1H), 4.21 (dd, 1H), 4.59 (dd,1H), 6.48-6.51 (brm, 1H), 6.62-6.67 (brm, 1H), 7.41-7.46 (brm, 1H), 7.78(dd, 1H), 8.37 (d, 1H), 8.43, (d, 1H), 8.65 (s, 1H). ³¹P NMR (500 MHz,D₂O): 17.47 (s).

Synthesis of (27)

5 mg of 6 in 200 μL of H₂O (pH adjusted to 8.3 with Na₂CO₃) was added to1 mg of Alexa Fluor® 647, succinimidyl ester (AF647, SE 26) in 50 μlanhydrous DMF, and the solution was stirred overnight. The solvent wasconcentrated under vacuum, and the resulting blue residue was dissolvedin 20% MeOH in 0.1 N TEAAc buffer (pH 5). The solution was purified bysemi-preparative HPLC under the following conditions: BeckmanUltrasphere C18 (250×10 mm) column, flow rate 4.0 mL/min of 0.1 N TEAAcbuffer (pH 5) for 5 min, linearly increasing to 40% of 70% MeOH in 0.1 NTEAAc buffer (pH 5) in 25 min, UV detection at 260 and 598 nm. Peakseluting at 17 min were collected as 27. The final amount of 27 isdetermined by UV absorption spectra (ε=240000 M⁻¹ cm⁻¹ at pH 7), and thesolution was lyophilized to yield blue-purple solids.

MS (positive ion ESI-MS gave the free acid molecular cation, calculated1198.2410 m/z, found 1197.1 m/z).

DISCUSSION

The inventors describe a new linking strategy centered on the couplingof a bisphosphonate or phosphonocarboxylate compound via a tertiary orheterocyclic nitrogen, such as the nitrogen of the pyridine ring in(1-hydroxy-2-pyridin-3-ylethane-1,1-diyl)bis(phosphonic acid) 1, toN-t-BOC-protected 1,2-epoxy-3-aminopropane 4 (easily prepared in 85%yield by conventional protection of commercially available allylaminewith tert-butoxycarbonyl anhydride, followed by epoxidation using MCPBA(31). The reaction proceeds under startlingly mild conditions: thebisphosphonate or phosphonocarboxylate compound and the oxirane reagentare stirred overnight in aqueous methanol at 35° C., resulting in clean,quantitative conversion to the drug-linker conjugate, which afterconventional deprotection of t-BOC with TFA, yields1-(3-amino-2-hydroxy-propyl)-3-(2-hydroxy-2,2-diphosphonoethyl)pyridiniumtrifluoroacetate (6). With slight modifications to these reactionconditions, N-alkylation of compounds such as[hydroxy(1H-imidazol-1-yl)methylene]bis(phosphonic acid) and{1-hydroxy-3-[methyl(pentyl)amino]propane-1,1-diyl}bis(phosphonic acid)have also been accomplished.

This remarkably facile reaction in water, generating the desired N+species, permits clean, quantitative ‘in situ’ attachment of drug-linkerto various moieties, such as fluorescent or near-IR labels or otheragents for various applications such as drug delivery systems, withoutthe need for any external reagent or catalyst. As such it should bebroadly applicable to functionalization of water-solublepyridyl-containing compounds, with (e.g. 1) or without therapeuticactivity (e.g. (1-hydroxy-2-pyridin-4-ylethane-1,1-diyl)bis(phosphonicacid)), and other tertiary or heterocylic nitrogen-containing compounds.Also advantageous is the formation of a secondary OH in the drug linkingstep, providing a hydrophilic group in mid-linker, which may aid inincreasing the aqueous solubility of the conjugate. This also gives riseto a chiral center at the C-2 of the linker, which may prove to be anintegral structural aspect for determining pharmacological properties ofdrug-like products.

Also key to this reaction is its regioselectivity. Chuiko et alpreviously reported O-alkylation of a bisphosphonic acid withdiethyl-oxiranylmethylamine in aqueous conditions near neutral pH withheating (60-70° C.) (6). However, the inventors discovered that at thistemperature, <10% O-alkylation and >90% N-alkylation of compound 1 wasobserved. By lowering the temperature of the reaction mixture to 40° C.,the inventors were able to afford N-alkylated analog of 1 cleanly.Generating O-alkylated analogs of bisphosphonates orphosphonocarboxylates may lower the parent compound's affinity to bone,thus adversely affecting key properties of the parent compound that maybe necessary to retain for their application as imaging probes.

After deprotection in TFA of analog 5, the unmasked amine 6 is reactedwith a mixture of 5- and 6-FAM isomers in activated succinimidyl form(5(6)-FAM, SE 7). The individual 5-(9) and 6-(10) isomers of theresulting fluorescently labeled drug analog, readily distinguished by ¹HNMR, are easily isolated by TLC followed by preparative reverse-phaseHPLC (9 is more mobile under the conditions chosen), or HPLC andsize-exclusion chromatography. To confirm the isomer assignment, thesynthesis and purification is repeated replacing the 5(6)-FAM, SE bypure 5-FAM, SE, which permitted unequivocal assignment of the ¹H NMRpeaks (particularly in the aromatic region) and HPLC elution order. The³¹P NMR spectra for the separated isomers are identical with a broadpeak at ˜16-17 ppm. The red-orange products are readily soluble inaqueous media near neutral or slightly basic pH. The UV-visible spectraare very similar in form to those of the parent 5(6)-FAM, dominated bythe major peak at 492 nm, apart from a small increase in molarabsorptivity near 260 nm, attributed to the presence of the pyridiniumchromophore of 1. The emission spectra of the FAM-labeled products inphosphate buffer, pH 7.2, show a maximum emission at ˜520 nm whereFAM-isomers exhibit about 75-80% relative quantum yields compared to5(6)-FAM. This slight decrease in fluorescence could be attributed tominimal exposure to light during work-up of the compounds or may be aninherent characteristic of the inventors' compounds.

TABLE 1 Characterization of the fluorescent labeled isomers 9 and 10Compound 9 10 ¹H NMR δ 3.37 (brt, 2H), 3.57 δ 3.27-3.37 (m, 2H), (dd, J= 14.1 Hz, 6.7 3.44 (dd, J = 14 Hz, 6.9 Hz, 1H), 3.61-3.69 (m, Hz, 1H),3.58 (dd, J = 1H), 4.22-4.31 (m, 1H), 14 Hz, 4.9 Hz, 1H), 4.36-4.45 (m,1H), 4.79 4.19 (brs, 1H), 4.35 (dd, (d, 1H), 6.61-6.71 (m, J = 14 Hz, J= 9.3 Hz, 4H), 7.11 (d, J = 9.2 1H), 6.60-6.73 (m, 4H), Hz, 2H), 7.33(d, J = 7.04 (d, J = 9.4 Hz, 8.0 Hz, 1H), 7.56 (s, 2H), 7.53 (s, 1H),7.81 0.14H), 7.83 (t, J = 7.2 (dd, J = 8.2 Hz, 6.4 Hz, Hz, 1H), 7.91 (d,J = 1H), 7.88 (d, J = 8.1 8.1 Hz, 1H), 8.15 (s, Hz, 1H), 7.95 (dd, J =1H), 8.44 (d, J = 8.3 8.0 Hz, 1.6 Hz, 1H), Hz, 1H), 8.56 (d, J = 8.43(d, J = 8.1 Hz, 5.9 Hz, 1H), 8.74 (s, 1H), 8.53 (d, J = 6.5 1H). Hz,1H), 8.70 (s, 1H). ³¹P NMR δ 16.47 (brs). δ 16.51 (brs). HRMS:positiveion Calculated 715.1089, Calculated 715.1089, MALDI (m/z) found715.1055. found 715.1082. UV λ_(max) (nm) 492 492 Emission λ_(max) (nm)523 520 HPLC retention time  44  27 (minutes)

A highly selective fluorescent probe derived from R-glycidol and 5-FAM,SE and 5(6)-FAM, SE, which also contained a benzenesulfonamide ligand tobind with high affinity to human carbonic anhydrase II (HCA II), waspreviously reported (4). Chen et al. attached the epoxide moiety (fromglycidol) to the fluorescent label via an ester bond, a linkage known tobe hydrolytically unstable under physiological conditions, andsubsequently conjugated the epoxide probe (in slight excess) to theheterocylic nitrogen of a histidine residue of HCA II (4).

In comparison, the current approach reverses the order of Chen (4) byfirst opening the epoxide by the heterocyclic (or tertiary) nitrogen,followed by conjugation to the imaging agent. This method generates amore hydrolytically stable amide bond between the label and the parentcompound. The labeled drug analogs 9 and 10 are stable in neutralconditions at room temperature for at least 24 hours by ¹H NMR andanalytical TLC (eluted with 100% MeOH, decomposition product will haveRf of 1.0). However, a similar drug analog that contained an ester bondbetween the drug and label showed decomposition by analytical TLC withina few hours under the same conditions.

Although the epoxide may first be attached to the imaging agent(preferably via an amide bond) and then conjugated to the heterocyclicor tertiary nitrogen, one must consider the cost of the imaging agentstarting material, which often varies but may be as high as ˜$900 for 5milligrams. Thus, synthesizing an imaging agent conjugated directly tothe epoxide (to be subsequently attached to the parent drug or compound)may be less cost efficient, especially in cases where a slight excess ofthe epoxide may be needed for N-alkylation reactions (4), thansynthesizing the N-alkylated analog of the parent compound first, whichcan then be conjugated to the commercially available imaging agents in a1:1 ratio.

At present, the individual 5- and 6-FAM, SE isomers are much moreexpensive than the commercially available isomer mixture, althoughseparation schemes based on derivativization have been recently proposed(26). In addition, separation of the isomers have reportedly beenachieved by reverse phase HPLC or by utilizing Biotage FLASH 75 system(which reportedly allows for gram scale separations) but slighthydrolysis of the succinimidyl esters was seen (1). However, theinventors found that separation of the isomeric product mixture was muchmore cost effective than either separation of starting material ester orsynthesis from pure isomers themselves.

Additionally, this type of technology provides for the synthesis ofphosphonates labeled with other fluorescent labels, such as AMCA-X andRhodamine Red-X, and near-infrared labels, such as Alexa Fluor® 647. Thecompounds are synthesized and purified similar to the method describedfor FAM-labeled products. The phosphonates are characterized by massspectrometry and NMR, UV absorption, and fluorescence emission spectra.

TABLE 2 UV absorption and fluorescence emission spectra of labeledcompounds. Compound Absorption λ_(max) (nm) Emission λ_(max) (nm)5(6)-FAM 492 516  8 493 518  9 493 523 10 493 520 17 493 520 18 493 52219 493 516 20 493 518  23* 567 589  24* 567 587  25** 346 —   27*** 648666 All UV spectra taken on DU 800 spectrophotometer and emissionspectra taken on Jobin Yvon Horiba FluoroMax-3 fluorometer. Samples in0.1 or 0.05 N phosphate buffer, pH 7.0; excitation wavelength at 490 nmunless otherwise noted. *Samples in 0.1 N phosphate buffer, pH 7.5;excitation wavelength at 520 nm. **Sample in 0.01 N phosphate buffer, pH7.5. ***Sample in 0.1 N phosphate buffer pH 7.0; excitation wavelengthat 600 nm.

Obviously, many modifications and variation of the invention ashereinbefore set forth can be made without departing from the spirit andscope thereof and therefore only such limitations should be imposed asare indicated by the appended claims.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

REFERENCES

The following references are cited herein. The entire disclosure of eachreference is relied upon and incorporated by reference herein.

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1. A compound of the formula: CH₂[O]CH(CH₂)_(n)R₁, comprising a C3 orlonger alkyl chain where one end is an oxirane that can be conjugated toa compound or drug, and the other, a group R₁ that can be conjugated toa second compound; and wherein the resulting compound may be used tolink a compound or drug to another drug, modify a moiety, link acompound or drug to a bead, link a compound or drug to a support, orlink a compound or drug to an imaging label.
 2. The compound of claim 1wherein the tertiary oxirane carbon is racemic.
 3. The compound of claim1 wherein the tertiary oxirane carbon is substantially pure as and R orS isomer.
 4. The compound of claim 1 wherein n is 1-12 and R₁ is anamino, hydroxyl, halogen, or other reactive group.
 5. The compound ofclaim 1 wherein n is 1 and R₁ is NH2.
 6. The compound of claim 1 whereinn is 1 and R₁ is a protected amino group.
 7. The compound of claim 1wherein n is 1 and R₁ is NHtBOC.
 8. The compound of claim 1 wherein n is2 and R₁ is an oxirane group.
 9. A method of synthesizing a linkablecompound comprising reacting the compound of claim 1 with a secondcompound containing a group capable of reacting with an oxirane.
 10. Themethod of claim 9 wherein n is 1 and R₁ is NHtBOC, and the tBOCprotecting group is removed after formation of the conjugate by reactionwith trifluoroacetic acid in water, aqueous DMF, or aqueous MeOH.
 11. Amethod of preparing the compound of claim 1 comprising: (a) reacting analkene comprising CH₂═CH—(CH2)_(n)R₁ with a reagent to protect R₁ and;(b) oxidizing the alkene group to an oxirane using meta-chloroperbenzoicacid.
 12. The method of claim 11 wherein n is 1 and R₁ is NHtBOC.
 13. Amethod of preparing a linker conjugate compound comprising: (a)dissolving a first compound of formula: CH₂[O]CH(CH₂)nR₁ in a solvent;and (b) reacting a second compound containing an amino group with saidfirst compound; wherein n is 1 and R₁ is NHtBOC of said first compound,and wherein said NHtBOC is dissolved in a solvent.
 14. The method ofclaim 13 wherein said NHtBOC dissolved in a solvent comprising MeOH orDMF.
 15. The method of claim 13 wherein said second compound comprises apyridyl group or other nitrogen-containing heterocyclic group, ortertiary amino group.
 16. The method of claim 15 wherein said secondcompound is a methylenebisphosphonate or phosphonocarboxylate. 17-31.(canceled)